A targetable ‘rogue’ neutrophil-subset, [CD11b+DEspR+] immunotype, is associated with severity and mortality in acute respiratory distress syndrome (ARDS) and COVID-19-ARDS

Neutrophil-mediated secondary tissue injury underlies acute respiratory distress syndrome (ARDS) and progression to multi-organ-failure (MOF) and death, processes linked to COVID-19-ARDS. This secondary tissue injury arises from dysregulated neutrophils and neutrophil extracellular traps (NETs) intended to kill pathogens, but instead cause cell-injury. Insufficiency of pleiotropic therapeutic approaches delineate the need for inhibitors of dysregulated neutrophil-subset(s) that induce subset-specific apoptosis critical for neutrophil function-shutdown. We hypothesized that neutrophils expressing the pro-survival dual endothelin-1/VEGF-signal peptide receptor, DEspR, are apoptosis-resistant like DEspR+ cancer-cells, hence comprise a consequential pathogenic neutrophil-subset in ARDS and COVID-19-ARDS. Here, we report the significant association of increased peripheral DEspR+CD11b+ neutrophil-counts with severity and mortality in ARDS and COVID-19-ARDS, and intravascular NET-formation, in contrast to DEspR[-] neutrophils. We detect DEspR+ neutrophils and monocytes in lung tissue patients in ARDS and COVID-19-ARDS, and increased neutrophil RNA-levels of DEspR ligands and modulators in COVID-19-ARDS scRNA-seq data-files. Unlike DEspR[-] neutrophils, DEspR+CD11b+ neutrophils exhibit delayed apoptosis, which is blocked by humanized anti-DEspR-IgG4S228P antibody, hu6g8, in ex vivo assays. Ex vivo live-cell imaging of Rhesus-derived DEspR+CD11b+ neutrophils showed hu6g8 target-engagement, internalization, and induction of apoptosis. Altogether, data identify DEspR+CD11b+ neutrophils as a targetable ‘rogue’ neutrophil-subset associated with severity and mortality in ARDS and COVID-19-ARDS.

Relevant to ex vivo analysis, these observations indicate that EDTA-anticoagulated blood exhibit less susceptibility to ex vivo experimental changes with increases in time and temperature (Fig. 1E, Supplementary Fig. S1C). For quantitative ex vivo ARDS patient sample analysis, we therefore used only EDTA anti-coagulated whole blood processed within 1-h from sampling from hereon, in order to minimize confounders that increase DEspRexpression ex vivo. This will avoid overestimating actual circulating levels in patient samples and false positives.

Detection of DEspR+ neutrophils in ARDS and COVID-19-ARDS lung tissue-sections.
To determine whether DEspR+ neutrophils are present in ARDS patient lung tissue, we performed immunohistochemistry analyses of post-mortem serial lung-tissue sections from patients with ARDS (n = 8) in regions of diffuse alveolar damage ( Fig. 2A-I) as well as, in areas of alveolar-capillary injury (Fig. 2J-K). Using an anti-DEspR mouse-recombinant mAb of hu6g8, hu6g8-m, immunohistochemistry with DAB chromogen (IHC-DAB) was optimized in order to track spatial patterns of DEspR+ expression in lung tissue-sections. This detected DEspR+ neutrophils in intrabronchiolar exudate, along with some DEspR(−) neutrophils identified by the characteristic polylobulated nuclei (Fig. 2B).
Furthermore, expression levels of all 9 genes representing DEspR's expression and functional network are significantly increased in COVID-19 compared to healthy controls (Fig. 3B), and in neutrophils compared to monocytes-macrophages in nasopharyngeal and broncho-lavage COVID-19 samples (Fig. 3C). The basis to study neutrophils is further supported by scRNA-seq documentation of increased expression of receptors to cytokines increased in ARDS 33 and/or in COVID-19-ARDS 34 , such as: interleukin (IL)-IL-6, IL-8, IL-1β, IL-18, and tissue necrotic factor-alpha (TNF-α) 35 (Supplementary Fig. S3G). These observations provide molecular evidence linking neutrophils as effectors of "cytokine storms" in ARDS, concordant with the known association of increased neutrophils with delayed apoptosis and mortality in ARDS 1,36,37 and COVID19-ARDS 38 .

Detection of DEspR+ CD11b+ neutrophil-subset in ARDS and COVID-19-ARDS.
To further study the DEspR+ neutrophil-subset, we completed a prospective pilot observational study of consented patients diagnosed with ARDS based on the Berlin Definition before the COVID19 pandemic. We prospectively studied consented ARDS patients (pre-COVID pandemic) regardless of the underlying acute disease trigger and associated comorbidities (Supplementary Table S1 for demographics).
First, we ascertained DEspR-specific immunotyping of whole blood samples from ARDS patients by validating our gating strategy for flow cytometry ( Supplementary Fig. S4A-E), DEspR-specific detection in duplicates (Supplementary Fig. S4F-I) and reproducibility in triplicates (Fig. 4A, Supplementary Fig. S4J). We note that the level of DEspR+CD11b+ neutrophil counts was not simply due to age in ARDS and COVID-19-ARDS (Supplementary Fig. S4K,L respectively).
Observing differential levels at the polar ends of the clinical spectrum of severity, we next stratified mortality outcomes in ARDS (Fig. 4F) and COVID-19-ARDS (Fig. 4G) patients by levels of DEspR+CD11b+ neutrophilcounts (K/μL whole blood). These pilot study trend-maps show an emerging differential pattern between survivors and non-survivors in ARDS and COVID-19-ARDS, providing bases for correlation analyses.
Association of DEspR+ CD11b+ neutrophil-subset with ARDS severity and mortality. To dissect the differential pattern emerging between survivors and non-survivors (Fig. 4F,G), we first performed correlation matrix analysis on a panel of DEspR-based flow cytometry markers, clinical markers of ARDS severity, and plasma biomarkers associated with neutrophil-mediated secondary tissue injury, and ET1 one of two DEspR ligands (Fig. 5A, Table 1). To assess clinical severity, we studied the number of ICU-free days at day 28 from ARDS diagnosis as a measure of mortality (death scored as [-1]) and speed to recovery within 28-days 39 , ARDS severity (SpO2/FiO2 or S/F ratio measure of hypoxemia), and Sequential Organ Failure Assessment (SOFA) scores on the day of sampling for flow cytometry analysis (t1-SOFA) and on day before ICU-discharge or ICU-death (t2-SOFA). To assess context in ARDS pathogenesis, we studied biomarkers of pathogenic events in ARDS relevant to neutrophil-mediated secondary tissue injury: interleukin-6 (IL-6 marker of cytokine storms), soluble C5b9 (terminal complex of complement activation), myeloperoxidase or MPO (neutrophil activation), ratio of the number of copies of mitochondrial to nuclear DNA in plasma (mtDNA-NET-formation) 24 , and DEspR+CD11b+ cytoplasts, (anuclear remnants of neutrophils associated with "vital NETosis") 25 .
Spearman rank correlation matrix analysis detected significant negative correlation between the number of DEspR+CD11b+ activated neutrophils and ICU-free days at day 28 (Fig. 5A, Table 1). Other DEspR-based FCMparameters, such as % of DEspR+CD11b+ neutrophils, monocytes and lymphocytes, also showed significant negative correlation with ICU-free days at day 28 (Table 1), in contrast with DEspR[-] neutrophil-counts which exhibited no significant correlation (Table 1). Spearman correlation analysis also detected significant correlation of number (#) and per cent (%) of DEspR+CD11b+ neutrophil with t2-SOFA scores but not with t1-SOFA (Fig. 5A, Table 1), supporting a pathogenic role in MOF-progression. In contrast, DEspR[-] neutrophil-counts, NLR, plasma levels of IL-6, MPO, and sC5b9 did not correlate with any of the measures studied (Fig. 5A, Table 1).

Association of DEspR+ CD11b+ neutrophils with COVID-19-ARDS severity and mortality. Sim-
ilarly, in COVID-19-ARDS pilot group, Spearman rank correlation matrix analysis showed significant, strong, negative correlation of DEspR+CD11b+ neutrophil-counts with ICU-free days at day 28 from ARDS diagnosis, and with ARDS severity S/F ratio, in contrast to no correlation with DEspR[-] neutrophil-counts (Fig. 5I, Table 2). Interestingly, the sum of %DEspR+ [monocytes and neutrophils] correlated with ICU-free days at day 28 with higher Spearman correlation coefficient, significance and power than either alone (Table 2). This observation is concordant with neutrophil-monocyte intravascular-interactions reported to contribute to systemic tissue injury in acute glomerular injury 40 . In contrast, the neutrophil lymphocyte ratio (NLR) showed significant albeit less robust correlation with ICU-free days at day 28 in COVID-19-ARDs compared to DEspR+CD11b+ neutrophil-counts. Comparative analysis of COVID-19-ARDS survivors and non-survivors showed significant differences in medians for S/F ratio ( Fig. 5J) but not for t1-SOFA score (Fig. 5K). Concordant with correlations detected, significant differences and large effect size were also detected between survivors and non-survivors for NLR (Fig. 5L), DEspR+CD11b+ neutrophil-counts (Fig. 5M), but not for DEspR[-] neutrophils (Fig. 5N).
To compare our pilot study observations with emerging biomarkers of severe COVID-19, we performed a retrospective analysis of COVID-19-ARDS patients requiring ventilatory support at Boston Medical Center. Data corroborate significant differences in NLR (Supplementary Fig. S5A-D), concordant with reports that increased NLR is an independent predictor of mortality in ARDS and COVID-19 41 . In contrast, C-reactive protein did not show significant differences between survivors and non-survivors (Supplementary Fig. S5E-H).

Characterization of DEspR+ NET-forming neutrophils in ARDS and COVID19-ARDS. To assess
NET-forming neutrophils relevant to detection of increased levels of NETs in in severe COVID-19 10,11,42,43 , we performed immunofluorescence staining to directly visualize and quantify NET-forming neutrophils in whole blood smears prepared from COVID-19-ARDS patients within 1-h from blood draw. Using high-resolution confocal imaging of immunofluorescent-staining for DEspR+CD11b+ expression, we detected differential levels of DEspR+CD11b+ NET-forming neutrophils in ARDS non-survivor, compared with ARDS-survivor and ICUpatient non-ARDS survivor (Fig. 6A).

Analysis of DEspR+ CD11b+ and DEspR+ CD66b+ NET-remnant cytoplasts and neutrophils.
Having detected DEspR+CD11b+ NET-forming neutrophils and DEspR+ MPO+ NETs on immunostained whole blood smears (Fig. 6A,B), we analyzed levels of cytoplasts as circulating NET-remnants by flow cytometry. We and another research site detected elevated DEspR+CD11b+ cytoplast levels in ARDS subjects (Fig. 6H) however, we did not observe association between circulating DEspR+CD11b+ cytoplast levels with clinical measures of ARDS severity (Fig. 5A, Table 1). Nevertheless, an independent small pilot study of patients with sepsis, and sepsis-ARDS confirmed elevated DEspR+CD66+ cytoplasts 44 and neutrophils in contrast to low to no levels in healthy donors ( Supplementary Fig. S6C-H). As a marker for neutrophil-degranulation, CD66b confirms neutrophil-derived cytoplasts. We note that neutrophils were isolated from whole blood samples via inertial microfluidic separation from RBCs 45 , performed however, ~ 3-h from blood draw-hence accounting for the much higher levels > 90% DEspR+ cytoplasts and neutrophils observed.

Ex vivo analysis of DEspR-inhibition in ARDS-patient neutrophils. To determine bioeffects of
DEspR-inhibition, we analyzed ARDS patient whole blood with humanized anti-DEspR IgG4 S228P antibody, hu6g8, added as ex vivo treatment for 17-20 h overnight with rotation to prevent aggregation. Controls comprised of patient-specific mock-treated and baseline pre-treatment controls (Fig. 7A). Comparative FCM-analysis showed that compared to baseline levels and after 17-20 h of ex vivo incubation at 37 °C, DEspR+ neutrophils increased in number compared with markedly decreased number of DEspR[-] neutrophils (Fig. 7A,B), suggesting that normal neutrophilic constitutive apoptosis is delayed in DEspR+ neutrophils but not in DEspR[−] neutrophils, and that some DEspR[−] neutrophils became DEspR+ with time.
To determine DEspR's role in delayed apoptosis, we inhibited DEspR via hu6g8-treatment, and performed flow cytometry after 17-20 h of treatment ex vivo. This showed that hu6g8 decreased the number of DEspR+ neutrophils in ARDS patient whole blood (Fig. 7B). Hu6g8 also reduced myeloperoxidase (MPO) (Fig. 7C) and soluble terminal complex of complement (sC5b9) (Fig. 7D) plasma levels, in contrast to greater than twofold increased levels in mock-treated controls, respectively after 17-20 h ex vivo incubation. These observations indicate that hu6g8 induced neutrophil apoptosis and function-shutdown of neutrophil-complement system reciprocal co-activation after 17-20 h of DEspR-inhibition via hu6g8-treatment. Importantly, neutrophil scRNAseq profile for CD47, the "don't eat me signal" is minimal, with only 0.3-0.91% of neutrophils with > 2 × fold CD47 (n = 19 COVID-19 patients) ( Supplementary Fig. S3F). This supports the therapeutic hypothesis that induction of apoptosis in DEspR+ neutrophils with no CD47 "don't eat me signal" can be cleared by efferocytosis.

Ex vivo analysis of DEspR-inhibition in Rhesus macaque neutrophils.
To further test that DEspRinhibition induces apoptosis in DEspR+ CD11b+ neutrophils, we performed live cell imaging of Rhesus macaque neutrophils exposed to fluorescently labeled hu6g8-AF568 or fluorescently labeled human IgG4-AF568 isotype control for 20 min at 4 °C to avoid non-specific endocytosis. We selected Rhesus neutrophils as model system since Rhesus-to-human neutrophils are more similar than human-to-mouse neutrophils 46 .
We first validated the presence of circulating DEspR+ CD11b+ neutrophils in Rhesus via flow cytometry using identical conditions to ex vivo analysis of ARDS patient samples ( Supplementary Fig. S7A-H). Next, we studied hu6g8 target engagement, internalization, and induction of characteristic apoptosis cell-budding bioeffects by confocal live cell imaging of Rhesus neutrophils. We exposed Rhesus neutrophils to either AF568-labeled anti-DEspR hu6g8 antibody (treatment) or IgG4-isotype (mock-treatment) control for 20 min at 4 °C to avoid non-specific endocytosis. After removing excess unbound antibody, 24-h live cell imaging was initiated with video-recordings. At t-45 min, live-cell images detected target engagement and internalization of hu6g8-AF568 antibody (Fig. 7E, Supplementary Fig. S7I) but not in the isotype control (Fig. 7F). Specificities were confirmed throughout with representative t-12 h timepoint images (Fig. 7G,H). At t-12 h, live cell imaging showed more apoptotic cell budding changes in NHP-neutrophils with internalized hu6g8 (Fig. 7G). In the isotype control, apoptotic cell budding was detected concordant with neutrophil constitutive apoptosis (Fig. 7H). SytoxGreen impermeable dye uptake marked loss of cell viability. Both cell death indicators increased with time (Fig. 7G,H).
At the 12-h midpoint, quantitation of apoptotic cell changes and SytoxGreen-positive non-viability were done. Quantitative analysis of 18 high power fields (HPFs) with 20-50 cells/HPF representing three independent experimental fields of view showed that hu6g8 induced apoptosis in DEspR+ neutrophils significantly greater than levels seen in isotype-treated control NHP cells (Fig. 7I). Importantly, hu6g8 induced apoptosis greater than constitutive apoptosis occurring in DEspR[-] cells unaffected by hu6g8 treatment (Fig. 7I). Interestingly, loss-ofviability staining by Sytox Green occurred in neutrophils not undergoing apoptotic cell budding, and was also   www.nature.com/scientificreports/ slightly greater in hu6g8-treated neutrophils compared with isotype mock-Tx controls (Fig. 7J), indicating that DEspR-inhibition may facilitate other programmed cell-death in neutrophils via decreased CIAP2 as observed in anti-DEspR mAb-treated pancreatic cancer stem cells 47 . Interestingly, shortly after the addition of SytoxGreen at t-15 min of live cell imaging, NET-formation with cytolysis was observed with DEspR+nucBlue+SytoxGreen+ NETs (Fig. 7K-M). DEspR+ neutrophils with internalized AF568-labeled hu6g8 were also observed (Fig. 7K). This contrasts t-12 h (Fig. 7G) where no NET-formation events were observed up to 24 h, suggesting the hypothesis that anti-DEspR induces neutrophil-apoptosis and pre-empts NET-formation. Lastly, NHP neutrophils shown to be > 90% DEspR+ at baseline flow cytometry analysis and day-1 live cell imaging (Fig. 7N) exhibited marked delayed apoptosis as live DEspR+ neutrophils were still observed 6-days after blood sampling (Fig. 7O).

Discussion
Data from multiple experimental systems among multi-center collaborators testing neutrophils in whole blood samples, either from NHVs, ARDS or COVID-19-ARDS patients, or Rhesus macaques, reproducibly identify the DEspR+ CD11b+ neutrophil-subset, concordant with cumulative evidence for neutrophil heterogeneity 1,48 . Detection of DEspR+ intracellular stores and increase in DEspR+CD11b+ neutrophils upon LPS-TLR4 activation in NHV blood samples indicate a dynamic subset-response to TLR4-activation. Importantly, the identification of the DEspR+CD11b+ neutrophil-subset in whole blood is supported by detection of DEspR+CD11b+ and DEspR+MPO+ neutrophils and monocyte/macrophages in postmortem lung tissue sections from ARDS and COVID-19-ARDS. Detection in the lung interstitium, intra-alveolar spaces, and vascular lumen in association with either diffuse alveolar damage (DAD) or alveolar-capillary injury confirm identification of the DEspR+ neutrophil-subset.
The direct visualization of DEspR+CD11b+ neutrophils with extruded DNA and intact nucleus and cell membrane in ARDS and COVID-19-ARDS patient whole blood smears match characteristics of NET-formation with mitochondrial DNA (mtDNA) and intact cell membranes 24,53 , and scanning electron microscopy images of NET-forming neutrophils 54 . Direct visualization of NET-forming neutrophils in patient blood smears documents intravascular events at time of blood draw, delineates NET-forming neutrophils as source of extruded DNA, and gives insight into pathophysiological context of different NET-subtypes. The algorithm-based automated quantitation of NET-forming neutrophils provides an objective quantitative method for direct morphological identification of NET-forming neutrophils in fixed patient blood smears, thus overcoming the limitation of nonquantitative visualization of IF-stained NETs 55 . These observations are supported by markedly elevated ratio of plasma mitochondrial DNA (mtDNA) to nuclear DNA (nDNA) copy-number in ARDS samples. Since elevated Table 1. Spearman rank correlation matrix analysis: ARDS. n = 19 subjects ARDS, all cause [pre-COVID19 pandemic]. CBC-differential NLR, neutrophil lymphocyte ratio calculated from ratio of absolute neutrophil to absolute lymphocyte counts. Flow cytometry parameters #DEspR+ CD11b+ Ns, total number (#) in K/μL of DEspR+ C11b + neutrophils (Ns); %DEspR + CD11b + Ns, % of DEspR + CD11b + neutrophils among total (CD11b+/−) neutrophils; %DEspR + CD11b + [Ns + Ms], sum of the % of DEspR + CD11b + neutrophils and monocytes; %DEspR + CD11b + Ms, %DEspR + CD11b + monocytes among total (CD11b+/−) monocytes; %DEspR+CD11b+ Ls, %DEspR+CD11b+ lymphocytes among total (CD11b+/−) lymphocytes; #DEspR(−) Ns, total number (#) in K/µL of DEspR(−) neutrophils (Ns). cytoplast HI , cytoplasts with high SSC or granularity; cytoplast LO , low SSC or low granularity cytoplast; Plasma biomarkers Plasma levels of ET1, endothelin-1, IL-6, interleukin-6; MPO, myeloperoxidase levels; sC5b9, soluble complement terminal C5b9-complex; and mt/nucl DNA, ratio of mitochondrial DNA copy number to nuclear DNA copy number. Clinical measures of ARDS severity ICU-free days by day 28 = [28 minus # ICU days] with NonSurvivors = [− 1] and Survivors > 28 ICU-days = 0; S/F ratio, SpO2/FiO2 ratio as a measure of hypoxemia severity; SOFA, Sequential Organ Failure Assessment score; t1-SOFA, SOFA score on day of flow cytometry analysis; t2-SOFA, SOFA score at end of ICU stay. Statistical analysis Spearman Rank Order Correlation coefficient rho (r) effect size: strong r 0.6-0.79; very strong r 0.8-1.0. Data points are peak values for subjects with multiple FCM analyses. Spearman Rank Correlation Coefficient (rho, r > 0.61, p < 0.05, has Power > 0.8 with n = 19; significant Spearman rho with power 0.8 highlighted in italics; significant Spearman rho but power < 0.8 in bold. www.nature.com/scientificreports/ mtDNA released by necrotic cells is observed in ARDS 56 and COVID-19-ARDS 57 , using mtDNA/nDNA ratio could help further dissect mtDNA-NET levels from cell necrosis which would exhibit a mtDNA/nDNA ratio < 1 given that mtDNA is less than 1% of nuclear DNA. Detection of the classic DEspR+MPO+ DNA-cloud, DEspR+CD11b+ NET-forming neutrophils with intact cell membranes, and long DNA-remnants with DEspR+CD11b+ microvesicles on the DNA strands in the same patient blood smear altogether indicate ongoing multiple intravascular NET-formation events in ARDS and COVID-19-ARDS. Multiple NET-subtypes are placed into pathophysiological context and are not due to confounders from different methods of analysis, sample source or procurement 53,58-60 . Relative pathophysiological relevance is supported by the significant correlation of intravascular DEspR+/CD11b+ NET-forming neutrophilcounts with multiple clinical measures of severity in COVID-19-ARDS, in contrast to non-correlation of NLR, IL-6, and sC5b9 61,62 in this prospective observational study.
Additionally, the presence of DEspR+CD11b+ ~ 200 micron-long intravascular DNA-strands, multi-NETforming neutrophil clusters, and NET-DNA strands straddling RBCs, are concordant with prior reports in COVID-19-ARDS and ARDS 1,10,11,60 . This combination of multiple intravascular NET-structures likely cause intravascular flow impedance leading to low-flow ischemia with or without micro-thromboses. Intravascular impedance to flow without microthrombi can account for persistence of low-flow or micro-ischemic events in different organs in severe ARDS and COVID-19-ARDS despite pharmacological thromboprophylaxis or antithrombotic treatment 63 .
Data showing that DEspR-inhibition leads to neutrophil apoptosis in ARDS patient and Rhesus macaque samples support DEspR as an actionable therapeutic target. Induction of apoptosis in dysregulated, apoptosisresistant neutrophil-subset(s) 64 implicated in progressive secondary tissue injury leading to ARDS-MOF 5 has been deduced to be a critical step towards initiation of resolution of excessive inflammation in ARDS 1 . Hence, targeted-inhibition of DEspR+ neutrophils with endpoint induction of neutrophil apoptosis presents a potential therapeutic approach with advantages. First, hu6g8-mediated induction of DEspR+ neutrophil apoptosis attains function-shutdown of neutrophil-complement reciprocal co-activation 65 , hence potentially breaking this self-sustaining pro-cell injury mechanism. Second, induction of DEspR+ neutrophil apoptosis without cell lysis provides a key step for efferocytosis 66 of the DEspR+ neutrophil subset. Thirdly, induction of apoptosis in DEspR+ neutrophils preempts progression to NET-formation, hence a potential therapeutic approach to preventing DEspR+ dysregulated NET-formation. This gains relevance when neutrophils are PADI4-negative, as Table 2. Spearman Rank Correlation Coefficients: COVID-19-ARDS subjects requiring ventilator support. n = 11 subjects with COVID-19-ARDS needing ventilatory support. CBC-differential NLR, neutrophil lymphocyte ratio. Flow cytometry parameters #DEspR+ CD11b+ Ns, total number (#) in K/μL of DEspR+C11b+ neutrophils (Ns); %DEspR+CD11b+Ns, % of DEspR+CD11b+ neutrophils among total (CD11b+/−) neutrophils; %DEspR+ CD11b+ [Ns+Ms], sum of the % of DEspR+ CD11b+ neutrophils and monocytes; %DEspR+CD11b+ Ms, %DEspR+CD11b+ monocytes among total (CD11b+/−) monocytes; #DEspR(-) Ns, total number (#) in K/µL of DEspR(−) neutrophils (Ns). Immunofluorescence Cytology parameters #DEspR+CD11b+ NET-forming Ns: number of DEspR+CD11b+ NET-forming Ns (% DEspR+ CD11b+NET-forming Ns x total number of DEspR+CD11b+ Ns); Circularity index, circularity index as a quantitative measure of NET-forming neutrophils with extruded DNA resulting in irregular perimeters with decreased similarity to a circle: outline of DAPI+ DNA fluorescence closest to 'perfect circle' = 1. Threshold 0.8: a NET-forming neutrophil has < 0.8 circularity index. Clinical measures of ARDS severity ICU-free days by day 28 = [28 minus # ICU days] with nonSurvivors = [− 1] and Survivors > 28 ICU-days = 0; S/F ratio, SpO2 converted to PaO2/FiO2 ratio as a measure of hypoxemia severity; SOFA, Sequential Organ Failure Assessment score; t1-SOFA, SOFA score on day of flow cytometry analysis; t2-SOFA, SOFA score at end of ICU stay (or day prior to death or discharge). Statistical analysis Spearman Rank Order Correlation coefficient rho (r) effect size: strong r 0.6-0.79; very strong r 0.8-1.0. Data points are peak values for subjects with multiple FCM analyses. Spearman Correlation Coefficient r > 0.76, alpha < 0.05, Power > 0.8, n = 11 (italics); Spearman r > 0.6, alpha < 0.05, power 0.7 to 0.8 (bold). www.nature.com/scientificreports/ seen in 98.6% of neutrophils in the COVID-19-ARDS RNA-seq files, hence non-responders to PADI4-inhibitors of NET-formation. While more studies are needed to elucidate mechanisms, induction of DEspR+ neutrophil apoptosis by DEspR inhibition complies with prior delineation that stopping neutrophil-mediated tissue injury requires induction of neutrophil apoptosis 1,64 . Importantly, consideration for potential on-target side effects, especially in the context of acute kidney injury as part of multi-organ failure in ARDS, highlights known DEspR+ expression in human medullary tubular epithelial cells 67 . In the presence of immunoglobinuria, anti-DEspR antibody passing through the glomerulus could present potential on-target tubular epithelial effects, but unlikely as antibody functionality will be altered in the increasingly acidic and hyperosmotic milieu in the kidney medullary lumen. Equally important, the critical limitation of pleiotropic neutrophil inhibitors in the context of ongoing infections and risk for secondary infections as in ARDS and COVID-19-ARDS, the target-specific inhibition of DEspR+ rogue neutrophils will spare DEspR[-]CD11b+ activated neutrophil subsets, hence preserve neutrophil defense functions against infections pertinent to critically ill patients with ARDS or COVID-19-ARDS.
Altogether, data identify the DEspR+CD11b+ neutrophil-subset as a therapeutic target with the potential to break the feed-forward progression of neutrophil-mediated tissue injury in ARDS and COVID-19-ARDS, while preserving DEspR[-] neutrophil functions. Data provide foundational basis for further study.
Limitations. We acknowledge the limitations of prospective pilot observational studies with n = 19 ARDS and n = 11 COVID-19-ARDS, and n = 19 COVID-19 scRNA profiles. With a focus on the study of neutrophils, we did not evaluate other cells with cytotoxic capabilities. We acknowledge the inherent limitations of the study of critically ill patients with limited patient samples for study, and limitations in our COVID-19-ARDS whole blood samples treated with 4% PFA to inactivate SARS CoV2 virus [final 2% PFA], leading to non-availability of plasma samples to perform ELISA studies on biomarkers and MPO-DNA complexes of NET-remnants.

Materials and methods
Study design. Different tasks in this interdisciplinary pilot observational study among different collaborators were compartmentalized in order to attain blinding of researchers during task-performance. The following tasks were compartmentalized: Study subjects. All subjects were identified in the ICU under study protocols approved by the Institutional Review Board (IRB) of Boston University (IRB H-36744). Each subject's legal authorized representative gave written informed consent for study participation in compliance with the Declaration of Helsinki.
We enrolled 19 ARDS patients in the pre-COVID-19 pandemic period, and 11 COVID-19 ARDS patients admitted to the intensive care unit (ICU) at Boston Medical Center. ARDS diagnosis was based on clinical diagnosis using the Berlin Definition. COVID-19 ARDS patients were ascertained as COVID-19 positive by SARS-CoV-2 PCR testing. Additional data were obtained prospectively from 16 COVID-19 ARDS patients to examine the time-course during ICU-hospitalization and correlation of other known markers with survival:  For study of ARDS and COVID-19-ARDS patient samples, whole blood (3 or 6 mls) was collected via preexisting indwelling peripheral vascular lines into K2-EDTA vacutainer tubes (FisherScientific, MA) from patients hospitalized in the ICU at Boston Medical Center by the ICU-nurse. COVID-19 patient EDTA-anticoagulated blood samples were immediately fixed with one volume of 4% PFA. Both Non-COVID and COVID-19 blood samples were processed for flow cytometry analysis within 1 h from blood collection. Platelet poor plasma was isolated and frozen at − 80 °C for future testing within 2 h from blood draw. Blood smears were prepared within 1 h from blood draw.

Flow cytometry analysis of blood samples [See Supplementary Methods for details.]
At BUSM, EDTA-anticoagulated blood samples from non-COVID ARDS subjects (100 μl per tube, × 2-3 replicates) were processed for flow cytometry within 1-h from blood sampling. [See Supplementary Methods for detail] Flow cytometry buffer comprised of Hank's balanced salt solution plus 2% heat-inactivated FBS as blocking agent; staining antibodies: 10 μg/ml of AF-647 labeled hu6g8 mAb, or the corresponding human IgG4-AF647 isotype IgG4, and 2.5 μg/ml anti-CD11b-AF488 or the corresponding mouse IgG1 kappa isotype control, AF-488; staining done at 4 °C × 30 min with rotation and protected from light; after staining, cells were fixed in 1% PBS-buffered PFA pH 7.4 at 4 °C, followed by RBC lysis at RT. After final wash, stained cells were resuspended in 400 μl HBSS 2% FBS, filtered and analyzed on a BD LSR-II flow cytometer. Analysis was done using FloJo Flow Cytometry Analysis Software (www. FloJo. com). Controls used were: both fluorescence minus one (FMO) controls, both isotype controls, compensation beads for both staining antibodies to check labeled antibody quality.
For disinfected COVID-19 blood samples (2%PFA-fixed), samples were washed 3 times with 8 volumes of HBSS + 2% FBS to remove residual fixative prior to processing for flow cytometry as described above. Each test sample run in duplicates.
At BWH, EDTA-anticoagulated whole blood samples were processed 2-3 h from sampling and white blood cells were separated from RBCs via Inertial Microfluidic Separation validated previously for neutrophil characterization 44   Representative image at t-12 h from video-recorded live cell imaging of isolated white blood cells (WBCs) documented to have > 90% DEspR+ CD11b+neutrophils among all neutrophils , and exposed to 10 μg/ml hu6g8-AF568 labeled antibody at 4 °C × 20 min to eliminate non-specific cell uptake by macropinocytosis or endocytosis. DEspR+ (red circle) Rhesus-neutrophils, apoptotic cell budding (encircled ○), and Sytox Green (SytoxG)-positive membrane permeable ( ). (H) Representative t-12 h image of isotype hu-IgG4 AF568 shows minimal to no isotype-AF568 (red circle) uptake; constitutive apoptosis cell budding (encircled ○), SytoxGreen-positive staining in cells with loss of cell membrane integrity (

Ex-vivo LPS treatment of human normal volunteer (HNV) neutrophils. [See Supplementary
Methods for details.] At Fraunhofer ITEM, heparinized whole blood was stored on ice until processing and used within 1-h after collection. Whole blood (100 µl) samples were washed with 1 ml of ice cold assay buffer, and cells were incubated in 100 µl of assay buffer containing bacterial endotoxin lipopolysaccharide LPS (100 ng/ml; Escherichia coli serotype 0111:B4) or assay buffer as control for 1 h at 37 °C. The reaction was then stopped, cells washed, then resuspended and cells were stained with hu6g8-PE (10 µg/ml) and CD11b-FITC for 30 min on ice under constant stirring in the dark. Cells were washed to remove unbound antibodies, fixed for 10 min at 4 °C, followed by RBC lysis. The cell pellet was resuspended in 250 µl flow cytometry buffer and was analyzed within 2 h using a Beckman Coulter Navios 3L 10C flow cytometer and data analyzed using Beckman Coulter Kaluza 2.1 Software.
To compare the levels of mitochondrial to nuclear DNA in human plasma samples we used the Nova-QUANT™ Human Mitochondrial to Nuclear DNA Ratio Kit (SIGMA-Aldrich cat# 72,620-1KIT) as per manufacturer's instructions. The kit measures the mtDNA copy number to that of nuclear DNA by Real-Time PCR of specific mitochondrial and nuclear genes optimized for equivalent amplification. Plasma DNA was isolated from 200 μl of plasma using the Quick-cfDNA Serum & Plasma Kit (Zymo Research, cat# D4076) as per manufacturer's instruction.
Immunofluorescence staining of NET-forming neutrophils. Blood smears were prepared by capillary action from EDTA anticoagulated whole blood (10 μL) samples on a Superfrost Plus Microscope slide (Fisher Scientific, cat# 12-550-15) within 1-h from blood sampling. Blood smears were air dried for 10 min then fixed with 100% Methanol (chilled to − 20 °C) for 10 min. Fixed slides were stored dry in − 20 °C freezer for future immunostaining. Immunofluorescence (IF)-staining to detect NET-forming neutrophils was done as described 68 , with custom modifications. We used anti-DEspR hu6g8 and anti-CD11b, as well as anti-DEspR and anti-MPO antibodies-conjugated to fluorophores (AF568, or AF488) for direct pair-wise immunostaining; DAPI for DNA detection. Chilled methanol fixation and permeabilization allowed fixation within 1 h from blood draw, eliminating need for paraformaldehyde and saponin. PBS with 5% FBS was used as blocking and binding solution for primary antibodies.
Fixed cell imaging of blood smears for quantiation of NET-forming neutrophils. Immunofluorescence imaging was performed as contract research service at Nikon Imaging Laboratory (Cambridge MA). Slides were imaged with a Nikon Ti2-E Widefield microscope equipped with a Plan Apo λ 20 × objective and Spectra LED light source and controlled by NIS-Elements. Briefly, an automated, JOBS routine in NIS-Elements Ex-vivo anti-DEspR treatment of ARDS patient blood samples. One ml of freshly obtained blood samples were incubated overnight at 37 °C with or without anti-DEspR mAb (hu6g8 at 100 μg/ml). After incubation half of the samples were subjected to FACS analysis as described above and the other half was processed for plasma isolation. Plasma MPO and C5b-9 levels were determined with corresponding ELISA kits as described above.